Embryonic stem cells (ESCs) are invaluable as models for embryonic development and how cell fate decisions are made. Post-transcriptional mechanisms including RNA binding proteins (RBPs) are major regulators of ESC differentiation and drive profound cell fate decisions during the continuum of the ESC-to-epiblast (EpiC) transition, the earliest differentiation event in the mammalian embryo. The RBP LIN41/TRIM71 is a major regulator of ESC development and cell fate decisions, is required for neural tube growth, and is associated with high grade/stage and poor outcome in human liver cancer. Thus the emerging importance of LIN41/TRIM71 in human development and disease warrants comprehensive analysis. As such, the full repertoire of transcripts regulated by LIN41/TRIM71 post-transcriptional repression has not been fully characterized. The main objectives of this proposal are 1) to characterize and dissect the biological roles of LIN41/TRIM71 in ESC differentiation, 2) to identify the global network of LIN41/TRIM71-bound mRNA sites of interaction, and 3) to build a global atlas of accessible RBP-bound sites in the ESC transcriptome during differentiation of LIN41 wildtype and knockout ESCs. Utilizing a combination of biochemical and computational techniques, we will characterize the global post-transcriptional program of this transition and gain insight into the interactions of known regulators (LIN41/TRIM71 and Argonaute2) as well as identify the molecular footprints of as-of-yet unknown post-transcriptional regulators within this context. The Blelloch lab has generated an in vitro reporter system in which a highly specific change in cell state representing the ESC-to-EpiC transition can be directly monitored. This research aims to build a broad map of LIN41/TRIM71-bound sites and coordinated networks of all RBPs active over the continuum of ESC differentiation, thus allowing a better understanding of the complex processes of early embryogenesis, developmental biology, and developmental disorders. We hypothesize that the absence of LIN41/TRIM71 will affect the G1/S transition by G1 phase elongation and accelerated ESC differentiation. Additionally, it is likely that identification of all potential RB bound sites in the presence versus absence of LIN41/TRIM71 and Argonaute by our unique method will show key changes in developmental-specific molecular footprints of these RBPs and the identify novel RBPs and previously unknown downstream targets important in alteration of ESC cell cycle at the G1/S transition. This proposal will for the first time build a broad temporal map of LIN41/TRIM71 targets in the ESC transcriptome during the ESC-to-epiblast transition. The results of this proposal will be relevant to the study of regulatory networks that control cell fate transitions in other cell types from proliferation to differentiation to neoplasi, and can potentially identify novel modulators of these processes. Completion of the work in this proposal will foster the fluid integration of genomic, computational, biochemical tools, and technical skills essential for the work I would like to do as an independent scientist.